3D-Shaped Binders of Unfolded Proteins Inducing Cancer Cell-Specific Endoplasmic Reticulum Stress In Vitro and In Vivo.

The amount of unfolded proteins is increased in cancer cells, leading to endoplasmic reticulum (ER) stress. Therefore, cancer cells are sensitive to drugs capable of further enhancing ER stress. Examples of such drugs include the clinically approved proteosome inhibitors bortezomib and carfilzomib. Unfortunately, the known ER stress inducers exhibit dose-limiting side effects that justify the search for better, more cancer-specific drugs of this type. Herein, we report on FeC 2, which binds to unfolded proteins prevents their further processing, thereby leading to ER stress and ROS increase in cancer cells, but not in normal cells. FeC 2 exhibits low micromolar toxicity toward human acute promyelocytic leukemia HL-60, Burkitt's lymphoma BL-2, T-cell leukemia Jurkat, ovarian carcinoma A2780, lung cancer SK-MES-1, and murine lung cancer LLC1 cells. Due to the cancer-specific mode of action, 2 is not toxic in vivo up to the dose of 147 mg/kg, does not affect normal blood and bone marrow cells at the therapeutically active dose, but strongly suppresses both primary tumor growth (confirmed in Nemeth-Kellner lymphoma and LLC1 lung cancer models of murine tumor) and spreading of metastases (LLC1).

Inhibition of heat-induced protein aggregation by zirconium phthalocyanines.

Specific proteins found in food sources tend to aggregate into fibrils under heat treatment; studying these aggregation processes and developing tools to control protein heat-induced aggregation is an active area of research. Phthalocyanine complexes are known to exhibit antiprionic and anti-fibrillogenic activity. Thus, the anti-fibrillogenic effect of a series of Zr phthalocyanines with different out-of-plane coordinated ligands, namely positively charged (PcZrLys2 ), negatively charged (PcZrCitr2 ), and group able to form disulfide bridges (PcZrS2 ), on the heat-induced aggregation of such proteins as BLG, insulin, and lysozyme was studied. The inhibition of reaction activity up to about 90% was observed in the presence of these compounds for all proteins. The effective concentration of the inhibitor was calculated for the compound with the highest activity (PcZrS2 ) to be 10.6 ± 3.6 and 7.3 ± 1.2 µM/L, respectively. Fluorescence spectroscopy studies demonstrated similar binding constants of three phthalocyanines binding with BLG globule. This is consistent with the results of molecular dynamics simulation, which imply the interaction of the globule with the tetrapyrrole macrocycle of phthalocyanine, leading to the globule stabilization. At the same time, TEM shows that in the presence of phthalocyanine PcZrS2 , thinner and longer fibrils were formed compared to control in all three proteins (BLG, insulin, and lysozyme). Thus, we can conclude that phthalocyanine PcZrS2 affects the amyloid aggregation's general mechanism, which is typical for proteins of different structures. Therefore, the phthalocyanine PcZrS2 is proposed as an anti-amyloidogenic agent suppressing heat-induced aggregation of proteins of different structures, making it potentially suitable for application in the food industry.

Sensing of Proteins by ICD Response of Iron(II) Clathrochelates Functionalized by Carboxyalkylsulfide Groups.

Recognition of elements of protein tertiary structure is crucial for biotechnological and biomedical tasks; this makes the development of optical sensors for certain protein surface elements important. Herein, we demonstrated the ability of iron(II) clathrochelates (1-3) functionalized with mono-, di- and hexa-carboxyalkylsulfide to induce selective circular dichroism (CD) response upon binding to globular proteins. Thus, inherently CD-silent clathrochelates revealed selective inducing of CD spectra when binding to human serum albumin (HSA) (1, 2), beta-lactoglobuline (2) and bovine serum albumin (BSA) (3). Hence, functionalization of iron(II) clathrochelates with the carboxyalkylsulfide group appears to be a promising tool for the design of CD-probes sensitive to certain surface elements of proteins tertiary structure. Additionally, interaction of 1-3 with proteins was also studied by isothermal titration calorimetry, protein fluorescence quenching, electrospray ionization mass spectrometry (ESI-MS) and computer simulations. Formation of both 1:1 and 1:2 assemblies of HSA with 1-3 was evidenced by ESI-MS. A protein fluorescence quenching study suggests that 3 binds with both BSA and HSA via the sites close to Trp residues. Molecular docking calculations indicate that for both BSA and HSA, binding of 3 to Site I and to an "additional site" is more favorable energetically than binding to Site II.

Induced CD of iron(ii) clathrochelates: sensing of the structural and conformational alterations of serum albumins.

An ability of inherently achiral macrobicyclic metal complexes iron(ii) clathrochelates to acquire an induced CD (ICD) output in the visible spectral range upon interaction with bovine serum albumin (BSA) was recently discovered. In the present work, the CD-reporting properties of iron(ii) clathrochelates to proteins and the thermodynamic parameters of their binding to albumins are evaluated. It is shown that iron(ii) clathrochelates functionalized by six ribbed carboxyphenylsulfide groups are able to discriminate between serum albumins of relative structure (here human and bovine albumins) by giving distinct ICD spectra. Besides, by the variation of the shape and intensity of CD bands, these cage metal complexes reflect the pH-triggered alterations of the tertiary structure of albumins. The constitutional isomerism (ortho-, meta- or para-isomers) of terminal carboxyphenylsulfide groups of iron(ii) clathrochelates strongly affects both the character of their ICD output upon binding with proteins and the parameters of the formed guest-host associates. Using isothermal titration calorimetry, it was determined that cage metal complexes bearing meta- and ortho-isomers of carboxyphenylsulfide groups possess higher association constants (Ka ∼ 2 × 104 M-1) and clathrochelate-to-BSA binding ratios (n = 2) than the para-isomer (Ka ∼ 5 × 103 M-1, n = 1). The iron(ii) clathrochelates are suggested to be potential molecular three-dimensional scaffolds for the design of CD-sensitive reporters able to recognize specific elements of protein surfaces.

Dicarboxyl-terminated iron(ii) clathrochelates as ICD-reporters for globular proteins.

Cage metal complexes iron(ii) clathrochelates, which are inherently CD silent, were discovered to demonstrate intensive output in induced circular dichroism (ICD) spectra upon their assembly to albumins. With the aim to design clathrochelates as protein-sensitive CD reporters, the approach for the functionalization of one chelate α-dioximate fragment of the clathrochelate framework with two non-equivalent substituents was developed, and constitutional isomers of clathrochelate with two non-equivalent carboxyphenylsulfide groups were synthesized. The interaction of designed iron(ii) clathrochelates and their symmetric homologues with globular proteins (serum albumins, lysozyme, ß-lactoglobulin (BLG), trypsin, insulin) was studied by protein fluorescence quenching and CD techniques. A highly-intensive ICD output of the clathrochelates was observed upon their association with albumins and BLG. It was shown that in the presence of BLG, different clathrochelate isomers gave spectra of inverted signs, indicating the stabilization of opposite configurations (Λ or Δ) of the clathrochelate framework in the assembly with this protein. So, we suggest that the isomerism of the terminal carboxy group determined preferable configurations of the clathrochelate framework for the fixation in the protein binding site. MALDI TOF results show the formation of BLG-clathrochelate complex with ratio 1 : 1. Based on the docking simulations, the binding of the clathrochelate molecule (all isomers) to the main BLG binding site (calyx) in its open conformation is suggested. The above results point that the variation of the ribbed substituents at the clathrochelate framework is an effective tool to achieve the specificity of clathrochelate ICD reporting properties to the target protein.

Induced chirality of cage metal complexes switched by their supramolecular and covalent binding.

An ability of the ribbed-functionalized iron(ii) clathrochelates to induce a CD output in interactions with a protein, covalent bonding or supramolecular interactions with a low-molecular-weight chiral inductor, was discovered. The interactions of CD inactive, carboxyl-terminated iron(ii) clathrochelates with serum albumin induced their molecular asymmetry, causing an appearance of strong CD signals in the range of 350-600 nm, whereas methyl ester and amide clathrochelate derivatives remained almost CD inactive. The CD spectra of carboxyl-terminated clathrochelates on supramolecular interactions or covalent bonding with (R)-(+)-1-phenylethylamine gave a substantially lower CD output than with albumin, affected by both the solvent polarity and the isomerism of clathrochelate's ribbed substituents. In supramolecular assemblies, the bands were most intensive for ortho-substituted carboxyl-terminated clathrochelates. The ortho- and meta-phenylethylamide cage complexes in tetrachloromethane inverted the signs of their CD bands compared with those in acetonitrile. It was suggested that the tris-dioximate metal clathrochelates possess a Russian doll-like molecular system. Because of the distorted TP-TAP geometry, their coordination polyhedron had no inversion centre and possessed an inherent chirality together with the equiprobability of its left(Λ)- and right(Δ)-handle twists. The selective fixation of one of these C3-distorted conformations resulted in the appearance of the CD signal in the range of their visible metal-to-ligand charge transfer bands. Calculations by DFT methods were used to illustrate the possible conformations of the macrobicyclic molecules, as well as the intramolecular interactions between the cage framework and optically active distal substituents responsible for the chirality induction of the metal-centred coordination polyhedra.